Showing papers by "Isak S. Pretorius published in 1988"
TL;DR: Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.
Abstract: Chromosomes of two closely related yeast strains, the amylolytic Saccharomyces diastaticus and the non-amylolytic Saccharomyces cerevisiae, were resolved by pulsed field gel electrophoresis (PFGE) and orthological field alteration gel electrophoresis (OFAGE). Electrophoretic karyotypes of these two strains are identical. Sixteen cloned Saccharomyces genes of known chromosomal location were used to identify individual chromosomes by Southern hybridization analyses. The Southern blots were reprobed with a cloned fragment of the STA2 glucoamylase gene of S. diastaticus. STA2 exhibits homology to STA1 and STA3 as well as the sporulation-specific glucoamylase (SGA) gene from both Saccharomyces strains. The three unlinked, homologous genes, STA1 (DEX2, MAL5), STA2 (DEX1) and STA3 (DEX3) encoding the extracellular glucoamylase isozymes GAI, GAII and GAIII in S. diastaticus were then assigned to chromosomes IV, II and XIV, respectively. The SGA gene, encoding an intracellular glucoamylase in both S. diastaticus and S. cerevisiae, was assigned to chromosome IX. Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) (STA10, INH1 and/or IST2) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.
31 citations
TL;DR: The successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals is reported on, indicating effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.
Abstract: A recombinant plasmid, pSR11.3, containing the α-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces strains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM277315B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified α-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the α-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 °C, respectively. Amylose hydrolysis profiles of the α-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.
26 citations